Hindered diffusion through an aqueous pore describes invariant dye selectivity of Cx43 junctions

The permselectivity (permeance/conductance) of Cx43-comprised gap junctions is a variable parameter of junctional function. To ascertain whether this variability in junctional permselectivity is explained by heterogeneous charge or size selectivity of the comprising channels, the permeance of individual Cx43 gap junctions to combinations of two dyes differing in either size or charge was determined in four cell types: Rin43, NRKe, HeLa43, and cardiac myocytes. The results show that Cx43 junctions are size- but not charge-selective and that both selectivities are constant parameters of junctional function. The consistency of dye selectivities indicates that the large continuum of measured junctional permselectivities cannot be ascribed to an equivalent continuum of individual channel selectivities. Further, the relative dye permeance sequence of NBD-M-TMA approximately Alexa 350 > Lucifer yellow > Alexa 488 > Alexa 594 (Stokes radii of 4.3 A, 4.4 A, 4.9 A, 5.8 A, and 7.4 A, respectively) and the conductance sequence of KCl > TEACl approximately Kglutamate are well described by hindered diffusion through an aqueous pore with radius approximately 10 A and length 160 A. The permselectivity and dye selectivity data suggest the variable presence in Cx43-comprised junctions of conductive channels that are either dye-impermeable or dye-permeable.     

Discovery and optimization of potent and selective benzonaphthyridinone analogs as small molecule mTOR inhibitors with improved mouse microsome stability

Starting from small molecule mTOR inhibitor Torin1, replacement of the piperazine ring with a phenyl ring resulted in a new series of mTOR inhibitors (as exemplified by 10) that showed superior potency and selectivity for mTOR, along with significantly improved mouse liver microsome stability and a longer in vivo half-life.     

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.     

Reduced neurogenesis in the rat hippocampus following high fructose consumption

In this study, we investigated how prolonged consumption of sugar solution affects hippocampal neurogenesis. We gave rats sucrose or fructose solution for four weeks and observed a 40% reduction in BrdU/NeuN-immunoreactive cells in the hippocampal dentate gyrus. This reduction in hippocampal neurogenesis was accompanied by increased apoptosis in the hippocampus and increased circulating levels of TNF-α. Therefore, we hypothesize that the reduction in hippocampal neurogenesis may be due to the increased apoptosis induced by TNF-α. Our results suggest that chronic ingestion of fructose is detrimental to the survival of newborn hippocampal neurones. The results presented in the present study add to the list of harmful effects associated with prolonged and excessive consumption of sugary beverages and soft drinks.     

Microenvironmental influence on pre-clinical activity of polo-like kinase inhibition in multiple myeloma: implications for clinical translation

Polo-like kinases (PLKs) play an important role in cell cycle progression, checkpoint control and mitosis. The high mitotic index and chromosomal instability of advanced cancers suggest that PLK inhibitors may be an attractive therapeutic option for presently incurable advanced neoplasias with systemic involvement, such as multiple myeloma (MM). We studied the PLK 1, 2, 3 inhibitor BI 2536 and observed potent (IC50<40 nM) and rapid (commitment to cell death <24 hrs) in vitro activity against MM cells in isolation, as well as in vivo activity against a traditional subcutaneous xenograft mouse model. Tumor cells in MM patients, however, don't exist in isolation, but reside in and interact with the bone microenvironment. Therefore conventional in vitro and in vivo preclinical assays don't take into account how interactions between MM cells and the bone microenvironment can potentially confer drug resistance. To probe this question, we performed tumor cell compartment-specific bioluminescence imaging assays to compare the preclinical anti-MM activity of BI 2536 in vitro in the presence vs. absence of stromal cells or osteoclasts. We observed that the presence of these bone marrow non-malignant cells led to decreased anti-MM activity of BI 2536. We further validated these results in an orthotopic in vivo mouse model of diffuse MM bone lesions where tumor cells interact with non-malignant cells of the bone microenvironment. We again observed that BI 2536 had decreased activity in this in vivo model of tumor-bone microenvironment interactions highlighting that, despite BI 2536's promising activity in conventional assays, its lack of activity in microenvironmental models raises concerns for its clinical development for MM. More broadly, preclinical drug testing in the absence of relevant tumor microenvironment interactions may overestimate potential clinical activity, thus explaining at least in part the gap between preclinical vs. clinical efficacy in MM and other cancers.     

Spreading of Heterochromatin Is Limited to Specific Families of Maize Retrotransposons

Transposable elements (TEs) have the potential to act as controlling elements to influence the expression of genes and are often subject to heterochromatic silencing. The current paradigm suggests that heterochromatic silencing can spread beyond the borders of TEs and influence the chromatin state of neighboring low-copy sequences. This would allow TEs to condition obligatory or facilitated epialleles and act as controlling elements. The maize genome contains numerous families of class I TEs (retrotransposons) that are present in moderate to high copy numbers, and many are found in regions near genes, which provides an opportunity to test whether the spreading of heterochromatin from retrotransposons is prevalent. We have investigated the extent of heterochromatin spreading into DNA flanking each family of retrotransposons by profiling DNA methylation and di-methylation of lysine 9 of histone 3 (H3K9me2) in low-copy regions of the maize genome. The effects of different retrotransposon families on local chromatin are highly variable. Some retrotransposon families exhibit enrichment of heterochromatic marks within 800–1,200 base pairs of insertion sites, while other families exhibit very little evidence for the spreading of heterochromatic marks. The analysis of chromatin state in genotypes that lack specific insertions suggests that the heterochromatin in low-copy DNA flanking retrotransposons often results from the spreading of silencing marks rather than insertion-site preferences. Genes located near TEs that exhibit spreading of heterochromatin tend to be expressed at lower levels than other genes. Our findings suggest that a subset of retrotransposon families may act as controlling elements influencing neighboring sequences, while the majority of retrotransposons have little effect on flanking sequences.     

Retrograde vesicle transport in the Golgi

The Golgi apparatus is the central sorting and biosynthesis hub of the secretory pathway, and uses vesicle transport for the recycling of its resident enzymes. This system must operate with high fidelity and efficiency for the correct modification of secretory glycoconjugates. In this review, we discuss recent advances on how coats, tethers, Rabs and SNAREs cooperate at the Golgi to achieve vesicle transport. We cover the well understood vesicle formation process orchestrated by the COPI coat, and the comprehensively documented fusion process governed by a set of Golgi localised SNAREs. Much less clear are the steps in-between formation and fusion of vesicles, and we therefore provide a much needed update of the latest findings about vesicle tethering. The interplay between Rab GTPases, golgin family coiled-coil tethers and the conserved oligomeric Golgi (COG) complex at the Golgi are thoroughly evaluated.     

Mechanism of HIV-1 viral protein R-induced apoptosis

The paradigm of HIV-1 infection includes the diminution of CD4(+) T cells, loss of immune function, and eventual progression to AIDS. However, the mechanisms that drive host T cell depletion remain elusive. One HIV protein thought to participate in this destructive cascade is the Vpr gene product. Accordingly, we review the biology of the HIV-1 viral protein R (Vpr) an apoptogenic HIV-1 accessory protein that is packaged into the virus particle. In this review we focus specifically on Vpr's ability to induce host cell apoptosis. Recent evidence suggests that Vpr implements a unique mechanism to drive host cell apoptosis, by directly depolarizing the mitochondria membrane potential. Vpr's attack on the mitochondria results in release of cytochrome c resulting in activation of the caspase 9 pathway culminating in the activation of caspase 3 and the downstream events of apoptosis. Vpr may interact with the adenine nucleotide translocator (ANT) to prompt this cascade. The role of Vpr-induced apoptosis in HIV pathogenesis is considered.     

Sphingosine 1-phosphate (S1P) receptor subtypes S1P1 and S1P3, respectively, regulate lymphocyte recirculation and heart rate

Sphingosine 1-phosphate (S1P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S1P(1) knock-out and the unavailability of selective agonists or antagonists. A potent, S1P(1)-receptor selective agonist structurally unrelated to S1P was found to activate multiple signals triggered by S1P, including guanosine 5'-3-O-(thio)triphosphate binding, calcium flux, Akt and ERK1/2 phosphorylation, and stimulation of migration of S1P(1)- but not S1P(3)-expressing cells in vitro. The agonist also alters lymphocyte trafficking in vivo. Use of selective agonism together with deletant mice lacking S1P(3) receptor reveals that agonism of S1P(1) receptor alone is sufficient to control lymphocyte recirculation. Moreover, S1P(1) receptor agonist plasma levels are causally associated with induction and maintenance of lymphopenia. S1P(3), and not S1P(1), is directly implicated in sinus bradycardia. The sustained bradycardia induced by S1P receptor non-selective immunosuppressive agonists in wild-type mice is abolished in S1P(3)-/- mice, whereas S1P(1)-selective agonist does not produce bradycardia. Separation of receptor subtype usage for control of lymphocyte recirculation and heart rate may allow the identification of selective immunosuppressive S1P(1) receptor agonists with an enhanced therapeutic window. S1P(1)-selective agonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S1P(1) suggests that selective tools for the resolution of function across this broad lipid receptor family are now possible.